This AREA proposal is aimed at the isolation and characterization of novel components from the venom of Conus regius, a Western Atlantic cone snail species. Cone snails are marine mollusks that use venom to capture their prey. The components of the venom (conopeptides) elicit a wide range of neurophysiological responses and show great potential as therapeutic agents. In this proposal we will investigate the novel conopeptides components of the venom of C. regius. Conus regius is biogeographically unique and our preliminary data shows that it possesses a wide array of new components with distinct neuromodulatory characteristics. We are proposing to utilize a high-resolution chromatographic scheme, developed in our laboratory, to efficiently separate the complex venom of C. regius into single components. These single components, isolated in nanomolar quantities provided by the native venom, will be analyzed by novel biomolecular NMR techniques, also developed in our lab. These nondestructive techniques rely on the use of a high resolution/magic angle spinning probe operating at low temperatures with homonuclear and heteronuclear 2D-NMR experiments that use pulse field gradients and selective excitation. The NMR data will be complemented by the use fempto-molar level ESI-MS/MS techniques that will allow us to carry out NMR/MS sequencing of the heavily post-translationally modified conopeptides in a non-destructive fashion. These analyses will be done in conjunction with high throughput in vitro functional bioassays using fluorescent-based/metal ion-specific techniques on murine cortical cells. These assays will assess the neuromodulatory characteristics of the conopeptides from C. regius. We will generate a novel set of conopeptides, which in addition of their potential therapeutic value; they can be used as molecular probes that would allow for the determination of function and mechanisms of actions of neuronal receptors.